Botulinum neurotoxins (BoNTs), produced by the anaerobic bacterium Clostridium botulinum, are the most potent toxins known1. These toxins cause botulism, a severe disease in humans and animals. Botulism usually results from ingestion of contaminated food. The toxins are first absorbed in the digestive system, possibly through a form of transcytosis across epithelial cells that line the gastrointestinal tract. Once in the bloodstream, the toxins target and enter motor nerve terminals and block the release of acetylcholine at neuromuscular junctions (NMJs), causing flaccid paralysis and may lead to death due to respiratory failure1, 2. Botulism is a rare disease in humans and thus the general population has not been immunized against these toxins; this is one of the reasons that BoNTs are among the most dangerous potential bioterrorism threats3.
There are seven serotypes of BoNTs (BoNT/A to G)1, 2. Each toxin is composed of a light chain (˜50 kDa) and a heavy chain (˜100 kDa), connected through a disulfide bond1. The heavy chain mediates cell-entry, via receptor-mediated endocytosis, and translocation of the light chain across the endosomal membrane into the cytosol1. The light chain is a protease that cleaves target proteins in cells1. BoNT/A and E cleave the peripheral membrane protein SNAP-25 (synaptosomal-associated protein of 25 kDa); BoNT/B,D,F and G cleave the vesicle membrane protein synaptobrevin (Syb); BoNT/C cleaves both SNAP-25 and the plasma membrane protein syntaxin4-9. SNAP-25, syntaxin and Syb are collectively referred to as SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins. These three SNAREs assemble into a complex that mediates the fusion of synaptic vesicles with the plasma membrane10-12; cleavage of these proteins thus inhibits synaptic vesicle exocytosis and blocks the release of neurotransmitters. Because of their ability to inhibit synaptic transmission, BoNTs are used to treat a wide spectrum of medical conditions ranging from overactive muscle disorders to chronic pain13-17.
The extremely high efficacy of these toxins is not only due to their enzymatic activity, but also involves their ability to recognize and enter presynaptic nerve terminals with high affinity and specificity. Thus, a major focus of research has been to identify the neuronal receptors for BoNTs. A “double-receptor” hypothesis has been proposed, in which BoNTs recognize nerve terminals by binding to two components: a group of membrane glycosphingolipids called gangliosides, and specific protein receptors18.
Complex forms of gangliosides, called polysialiogangliosides (PSG), have been shown to bind BoNT/A, B and E with low affinity19-22. Cells lacking gangliosides are resistant to the binding and entry of BoNT/A, B and G; entry can be rescued by loading cell membranes with exogenous gangliosides23-25. Furthermore, mice lacking PSG showed decreased sensitivities to BoNT/A, B, C and G25-29. Interestingly, it was recently reported that BoNT/D does not interact with gangliosides and loss of PSG does not diminish the entry of BoNT/D into neurons27. Furthermore, mice lacking PSG exhibit the same sensitivity to BoNT/D as wild type (WT) mice, indicating that not all BoNTs utilize gangliosides as co-receptors27. It has not been reported whether gangliosides are essential for the entry of BoNT/E or BoNT/F into neurons.
Among the seven BoNTs, the protein receptors for BoNT/A, B and G have been identified (see e.g. U.S. patent application Ser. No. 10/695,577). Two homologous synaptic vesicle membrane proteins, synaptotagmins I and II (Syts I/II), were first found to bind BoNT/B30, 31 and were subsequently shown to function as the protein receptors that mediate entry of BoNT/B into cells25, 32. The toxin binding site lies in a short intravesicular region that is conserved between Syt I and II32. In addition, BoNT/G was also found to utilize Syt I/II as its receptor by recognizing the same toxin binding site on Syt I/II as BoNT/B25, 29, 33.
The co-crystal structure of BoNT/B bound to the toxin binding domain of Syt II was recently reported. This structure revealed that the toxin binds Syt II through a hydrophobic groove within the C-terminal region of BoNT/B24, 34. This hydrophobic groove is conserved in all subtypes of BoNT/B, as well as in BoNT/G24, 29, 34.
The receptor for BoNT/A was recently identified as another synaptic vesicle membrane protein, SV235, 36. All three isoforms of SV2 in mammals (SV2A, B and C) bind BoNT/A and mediate its entry into cells35. SV2 contains twelve transmembrane domains with one large luminal domain (the fourth luminal domain, L4) between the seventh and eighth transmembrane domains37-40. SV2 is a proteoglycan on synaptic vesicles and is heavily glycosylated, possibly through three putative N-glycosylation sites within the L4 luminal domain37, 38, 40-42. Interestingly, the BoNT/A binding site was mapped to a region within the SV2-L4 domain that contains two putative glycosylation sites35. It is not clear whether glycosylation of SV2 affects the binding of BoNT/A.
BoNT/E is one of four BoNTs (BoNT/A, B, E and rarely F) that are associated with human botulism43. It is also one of the leading causes of botulism outbreaks among wild fish and birds44. The protein receptor for BoNT/E, however, has not been identified.
Previous studies revealed that neuronal activity facilitated paralysis in diaphragm muscle preparations exposed to BoNT/E, and increased the cleavage of the substrate protein—SNAP-25—in cultured hippocampal neurons45, 46, providing indirect evidence that synaptic vesicle recycling may enhance the entry of BoNT/E. However, it was reported that BoNT/E does not bind to the recombinant luminal domains of Syt I/II or SV2 purified from E. coli32 33 35, 36.
Previously, BoNT/A and E was reported to bind Syt I in a ganglioside independent manner (Li and Singh, 1998, Isolation of synaptotagmin as a receptor for types A and E botulinum neurotoxin and analysis of their comparative binding using a new microtiter plate assay. J. Nat. Toxins. 7:215-26). This reported binding, however, turned out to be at best non-specific, as subsequent work could not confirm any significant binding between BoNT/E and Syt I (see Dong et al., 2003, Synaptotagmins I and II mediate entry of botulinum neurotoxin B into cells, J. Cell. Bio. 162:1293-1303, at FIG. 1A). The lack of binding between BoNT/E and Syt I has also been further confirmed by others (see Rummel et al., 2004, Synaptotagmins I and II Act as Nerve Cell Receptors for Botulinum Neurotoxin G, J. Biol. Chem. 279:30865-30870, at FIG. 1B).
There is thus a need to identify the protein receptor for BoNT/E and to determine whether gangliosides serve as co-receptors for this toxin. Identification of the receptor for BoNT/E will be extremely useful for designing molecules that can reduce or completely inhibit its toxicity. Similarly, knowledge of the BoNT/E binding domain of the receptor will allow the use of polypeptides containing the domain and peptidomimetics thereof as competitors for BoNT binding, thereby reducing or completely inhibiting BoNT toxicity.
There is also a need to target the enzymatic domain of BoNTs, i.e. the light chain that can cleave SNARE proteins, into non-neuronal cells. Many types of cells use SNARE proteins to mediate vesicle release of hormones, cytokines, etc. It is well-known that vesicle-mediated release of transmitters and hormones constitutes a fundamental means of intercellular communication and malfunction of this process leads to many diseases. BoNTs have proven to be a powerful tool to treat diseases caused by over-active neurons. Currently, however, one cannot use BoNTs to treat non-neuronal cells for excessive secretion, mainly because BoNTs cannot enter these cells which do not express BoNT receptors. Even if non-neuronal cells did express BoNT receptors, it was not known if BoNT would be effective in these cells, as non-neuronal cells are known to lack synaptic vesicle recycling pathway, but no entry pathway other than synaptic vesicle recycling was known to result in functional entry of BoNT.
There is a further need for non-neuronal cells who express a BoNT receptor. Such cells would be more stable and more easily to culture, and can be used to replace using primary culture neurons for studying toxin actions and screening toxin inhibitors.